Mechanisms that Govern Cardiomyocyte Proliferation and Remuscularization following Ventricular Injury

摘要 心室损伤后心肌细胞增殖和肌化的调控机制 总体方案 心肌梗死治疗的总体目标是替换缺血性损伤引起的瘢痕组织 有功能的心肌由于成年哺乳动物心肌细胞（CM）是非增殖性的， 心脏细胞治疗的移植率极低，大多数肌肉重建计划都是在 梗塞没有成功。然而，我们实验室最近的初步研究使用新生儿 猪已经表明，当在出生后第1天（P1）诱导心肌梗塞（MI）时，CM重新进入细胞 循环，增殖，并完全恢复心脏功能，几乎没有疤痕。此外，我们发现， 这些P1损伤的新生儿心脏具有非常活跃和延长的CM增殖机制， 随后在P28时发生第二次LAD结扎损伤，导致LAD后第2-7天发生大面积梗死（TTC 结扎，在损伤后4周没有产生可见的梗塞。这是一个值得注意的结果， 首次发现大型哺乳动物的心脏可以通过CM增殖使梗死的心脏组织肌化。 包括本计划项目补助金（PPG）申请的研究将检查CM 重新进入细胞周期和新的策略，以重建受损的心脏。项目1将确定CM细胞周期 在一日龄猪中由MI激活的调节因子，并构建人心肌补片（hCMP）， 来自具有活化细胞周期的增殖hiPSC-CM层的前所未有的临床相关尺寸 调节因子和其他心肌细胞;随后的实验将确定所识别的因子和 hCMPs可以使MI后的成年猪心脏肌肉化。项目2将使用遗传策略，病毒载体， 和修饰的RNA，以研究包括Gli 1在内的Sonic Hedgehog信号通路的成员是否 和Sox 4，已经显示在培养的CM中诱导增殖，将促进CM增殖 在成年老鼠和猪受伤的心脏中。此外，研究将审查主调节器的能力， Etv 2，以促进新生血管形成并促进成年小鼠和猪的受损心脏的修复。项目3 将是以前观察的延伸，即哺乳动物细胞周期停滞至少部分是由 出生后氧代谢增加，严重的全身性缺氧使脯氨酸上调 代谢和诱导CM增殖成年小鼠;拟议的研究将检查是否脯氨酸 代谢调节慢性缺氧时CM的存活和增殖。总的来说，这三个项目 相关的核心和研究人员的专业知识将加速和扩大研究，以解决 P01提案的中心目标：通过促进从“内部”重建受损心室， 内源性CM增殖，并从“外部”，通过移植功能成熟的hCMPs， 体内CM增殖。

ABSTRACT Mechanisms that govern cardiomyocyte proliferation and remuscularization following ventricular injury Program Overall The overall goal for the treatment of myocardial infarction is to replace the scar tissue caused by ischemic injury with functional cardiac muscle. Since adult mammalian cardiomyocytes (CMs) are non-proliferative, and the engraftment rate for cardiac cell therapy is extremely low, most of the remuscularizing initiatives following infarction have been unsuccessful. However, recent preliminary studies from our laboratories using neonatal pigs have shown that when myocardial infarction (MI) is induced on postnatal day 1 (P1), CMs re-enter the cell cycle, proliferate, and completely restore cardiac function with little scarring. Furthermore, we have found that these neonatal hearts with the P1 injury, have a very active and prolonged CM proliferative machinery, and consequently a second LAD ligation injury at P28, which resulted in a large infarct (TTC) at Day 2-7 post LAD ligation, produced no visible infarct 4 weeks following injury. This was a remarkable result as it demonstrated, for the first time, that a heart of large mammal could remuscularize infarcted heart tissue by CM proliferation. The studies comprising this Program Project Grant (PPG) application will examine mechanisms whereby CMs reenter the cell cycle and new strategies to remuscularize injured hearts. Project 1 will identify the CM cell-cycle regulators that are activated by MI in one-day-old pigs and construct human cardiac muscle patches (hCMP) of unprecedented clinically relevant dimensions from layers of proliferating hiPSC-CMs with activated cell cycle regulators, and other cardiac cells; subsequent experiments will determine whether the identified factors and hCMPs can remuscularize the hearts of adult pigs after MI. Project 2 will use genetic strategies, viral vectors, and modified RNAs to investigate whether members of the Sonic Hedgehog signaling pathway including Gli1 and Sox4, which have already been shown to induce proliferation in cultured CMs, will promote CM proliferation in the injured hearts of adult mice and pigs. In addition, studies will examine the capacity of the master regulator, Etv2, to promote neovascularization and promote repair of the injured hearts of adult mice and pigs. Project 3 will be an extension of previousobservations that mammalian cell-cycle arrest is at least partially induced by the increase in oxygen metabolismthat occurs after birth, and that severe systemic hypoxia upregulates proline metabolism and induces CM proliferation in adult mice; the proposed studies will examine whether proline metabolism regulates CM survivaland proliferation during chronic hypoxia. Collectively, these three projects, the associated cores and the expertise of the investigators will accelerate and amplify the studiesto address the central objective of this P01 proposal: To remuscularize the injured ventricle from “within,” by promoting endogenous CM proliferation, and from “outside,” bytransplanting functionally mature hCMPs that are primed for in-vivo CM proliferation.